Carcinogen-Binding Proteins in the Rat Ventral Prostate

[CANCER RESEARCH 46, 651-657, February 1986]

Carcinogen-binding Proteins in the Rat Ventral Prostate: Specific and Nonspecific High-Affinity Binding Sites for Benzo(a)pyrene, 3-Methylcholanthrene, and 2,3,7,8-Tetrachlorodibenzo-p-dioxin1

Peter Soderkvist,2 Lorenz Poellinger, and Jan-Ã‚ke Gustafsson

Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital F-69, S-141 86 Huddinge, Sweden

ABSTRACT cytochrome P-450-dependent microsomal enzyme activity AHH3 as well as Â¡mmunodetectable amounts of the major 0-naphtho- The polychlorinated dibenzodioxin [3H]-2,3,7,8-tetrachlorodi- flavone-inducible isozyme of cytochrome P-450, cytochrome P- benzo-p-dioxin (TCDD) and the carcinogens [3H]benzo(a)pyrene 450c (the form mainly responsible for AHH-activity) are very low. and [3H]-3-methylcholanthrene bound to saturable binding sites Following treatment with /3-naphthoflavone or TCDD, an approx imately 500-fold induction of cytochrome P-450c is detectable in cytosol from the rat ventral prostate. Analysis of equilibrium binding parameters in diluted cytosol preparations indicated an by both immunochemical techniques and AHH activity determi nations (3-7). Moreover, this induction in the prostate has been apparent Ka of ~2 nw and a binding capacity of approximately 1 nmol/mg cytosolic protein, corresponding to ~5% of the total shown to be accompanied by an increased capability to form mutagenic metabolites from the promutagens 2-aminofluorene protein content. However, gel permeation chromatography anal and B(a)P as determined by the Ames' Sa/mone//a/microsome ysis as well as velocity sedimentation analysis on sucrose gra dients of [3H]TCDD-labeled rat prostatic cytosol indicated binding assay (6). of [3H]TCDD to two discrete species. These analyses indicated A substantial amount of evidence indicates that the induction a sedimentation coefficient of 3.6-3.8S, a Stokes radius of 25- of cytochrome P-450c in several tissues is mediated by binding 28 A, and a calculated relative molecular weight of 42,000- of the inducing agent to an intracellular, soluble receptor protein, which in a poorly understood process leads to an increased 45,000 for the most abundant binding species. The other binding affinity of the ligand-receptor complex for target sites within the species sedimented at 4-5S under high ionic strength conditions and at 8-1 OS under low ionic strength conditions and had a cell nucleus (8). TCDD is one of the most potent agonists known today for both the enzyme-induction response (9) and receptor Stokes radius of approximately 60 A, a relative molecular weight of ~100,000, and an estimated concentration of 5-20 fmol/mg binding (10). Other agonists include such well-known carcino cytosolic protein. Binding of [3H]TCDD to this species was gens as 3-MC and B(a)P (10). The TCDD receptor has been displaceable by a 200-fold M excess of 2,3,7,8-tetrachlorodiben- demonstrated in several extrahepatic tissues in both rats and mice (11,12). Surprisingly, no TCDD receptor has been reported zofuran. Therefore, this species was tentatively identified as the TCDD receptor. The properties of the high-capacity binder of for the rat ventral prostate, possibly indicating a nonreceptor [3H]TCDD were found to be similar to the characteristics of a mediated induction process of cytochrome P-450c in this tissue. However, a very high degree of nonspecific (nonreceptor) binding protein previously purified from the rat ventral prostate, prostatic of [3H]TCDD to an unidentified component in undiluted cytosol secretory protein, which binds androgens as well as estramus- from the rat prostate has been reported (11,12). It has also been tine, a nitrogen mustard derivative of estradiol. The binding of reported that [3H]B(a)P readily interacts with components in rat estramustine to diluted prostatic cytosol was shown to be com petitively inhibited by 2,3,7,8-tetrachlorodibenzofuran. Moreover, prostatic cytosol (13). In this paper, we characterize the binding of various radioligands ([3H]TCDD, [3H]-3-MC, and [3H]B(a)P) to purified prostatic secretory protein bound [3H]TCDD, [3H]- benzo(a)pyrene, as well as [3H]-3-methylcholanthrene. It is sug ventral prostatic cytosol and fractions thereof. gested that binding to this protein is responsible for the high- binding capacity of carcinogens in cytosol from the rat ventral MATERIALS AND METHODS prostate. Chemicals. [1,6-3H]TCDD (28 Ci/mmol) was a generous gift from Dr. A. Poland (Madison, Wl). [G-3H]3-MC (27 Ci/mmol) and [G-3H]B(a)P (25 INTRODUCTION Ci/mmol) were from the Radiochemical Centre, Amersham, England. [1- 14C]-Glucose (8 Ci/mol) was from New England Nuclear Corp. (Boston, As a chemical etiology of prostatic cancer recently has been MA). Unlabeled TCDF was kindly supplied by Dr. C. Rappe (UmeÃ , suggested (1, 2), several studies have addressed the issue Sweden). Estramustine and [2,4,6,7-3H]estramustine (107 Ci/mmol) were whether the prostatic gland possesses enzymes necessary for generously provided by Leo Pharmaceuticals (Helsingborg, Sweden). the metabolic activation of chemical carcinogens. In the rat Activated charcoal (Norit A), cytochrome c (equine heart), albumin (bovine ventral prostate of untreated rats, it has been shown that the serum fraction V), rabbit IgG, and catalase (bovine liver) were purchased 3 The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; cytochrome P-450C, the major form of cytochrome P-450 isolated from rat liver microsomes of Received 4/24/85; revised 10/15/85; accepted 10/16/85. 0-naphthoflavone-(5,6-benzoflavone}-treated rats; TCDD, 2,3,7,8-tetrachlorodi- 1Supported by grants from the Swedish Medical Research Council (Grant 03X- benzo-p-dioxin; 3-MC, 3-methylcholanthrene; B(a)P, benzo(a)pyrene; TCDF, 6807), the Swedish Cancer Society, and the Swedish Board for Planning and 2,3,7,8-tetrachlorodibenzofuran; estramustine, 1,3,5(10)-estratriene-3,17/i-diol-3- Coordination of Research. N-bis(2-chloroethyl)carbamate; DCC, dextran-coated charcoal; PSP, prostatic ste 2 To whom requests for reprints should be addressed. roid binding protein; PAH, polycyclic aromatic hydrocarbon.

CANCER RESEARCH VOL. 46 FEBRUARY 1986 651 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. CARCINOGEN-BINDING PROTEINS IN THE RAT PROSTATE

from Sigma Chemical Co. (St. Louis, MO). Sephacryl S-300, Sephadex (61.5 A); catalase (51.3 A); bovine serum albumin (35.9 A); and cytc- G-25 and G-100 fine, DEAE-Sepharose, heparin-Sepharose, blue dextran chromec(17.9A). 2000, and dextran T-70 were from Pharmacia Fine Chemicals (Uppsala, Calculation of Hydrodynamic Properties. Relative molecular Sweden). All other chemicals were analytical grade products from either weights, and frictional ratios due to shape, f/f0, were calculated from the Sigma or Merck AG (Darmstadt, Federal Republic of Germany). Stokes radius, R5, and sedimentation coefficient as described by Siegel Animals. Male Sprague-Dawley rats weighing 200-300 g were used and Monty (22), assuming a partial specific volume of 0.725 cm3/g and throughout this study. a solvation factor of 0.2 g/g of solute (18). Axial ratios, a/b, for hydro- Buffer. The following buffer was routinely used: 20 rnw potassium dynamically equivalent prolate ellipsoids were derived from the values phosphate-1 HIM EDTA-2 rriM 2-hydroxyethylmercaptan-10% (w/v) glyc- listed by Schachman (23). S and fls values for the protein standards erol, pH 7.2. were obtained from the literature (24, 25). Preparation of Cytosol. Rats were killed by cervical dislocation and Heparin-Sepharose Chromatography. Heparin-Sepharose was equil the two ventral prostate lobes were immediately excised and removed ibrated in buffer and packed into a siliconized column (2.6 x 8 cm). onto ice. All successive work was carried out at 0-4Â°C unless indicated Nonlabeled cytosol was applied to the column which was then washed differently. The prostates were finely minced with a pair of scissors in with 2-3 column vol of buffer, whereafter retained material was eluted approximately 5-10 vol of buffer and thereafter homogenized in 2 vol of with 1 column vol of buffer containing 0.5 M NaCI at a flow rate of 4 ml/ buffer in a teflon/glass Potter-Elvehjem homogenizer. The homogenate cm2/h. was then centrifuged at 140,000 x gav for 45 min. The supernatant, Saturation Studies. Saturation studies were performed in siliconized hereafter termed cytosol, was removed and care was taken to avoid the glass tubes at cytosolic protein concentrations of approximately 1 ^g/ml floating lipid layer. buffer (26, 27) containing 0.1% (w/v) gelatin (26). Gelatin was included Purification of PSP. The mincing procedure of the prostatic gland in the incubation mixture in order to avoid adsorption of ligand or protein resulted in release of secretory fluid from the ductules and lumina (14) to the wall of the incubation tube. Under these conditions, up to 10 nw [3H]TCDD and approximately 25 nw [3H]B(a)P and [3H]-3-MC, respec which subsequently together with the cytosol was used for purification of PSP by anion-exchange and gel permeation chromatography as tively, could be dissolved. Diluted cytosol or purified PSP were incubated in 0.5-ml aliquots with varying concentrations of ligand at 37Â°C for 30 described by Heyns et al. (15). Purified PSP was quickly frozen in aliquots and stored at -70Â°C. In some cases, PSP was delipidated by precipi min. The tubes were removed onto ice, and unbound ligand was ad sorbed by the addition of 0.5 ml of an ice-cold DCC suspension [1.5% tation in acetone (16,17) before assessment of ligand binding. (w/v) charcoal-0.05% (w/v) dextran T-70-0.1% (w/v) gelatin in buffer]. In Vitro Labeling Conditions. For determination of TCDD receptor The incubations were agitated and left on ice for 10 min. The charcoal binding, crude cytosol or various Chromatographie fractions of cytosol were labeled with 3-5 nw [3H]TCDD at the indicated protein concentra was then pelleted by centrifugation at 1500 x g for 10 min, whereafter tions for 2-18 h at 0-4Â°C, usually in the absence or presence of a 200- the supernatant was assayed for radioactivity. To determine the total radioactivity added, aliquots (generally, 20 >solubility in analyzed by Scatchard plots (28) or double-reciprocal plots using linear water and similar affinity for the receptor (10). Bound and free ligand regression analysis to determine apparent equilibrium parameters of were usually separated by DCC adsorption [1.9% (w/v) charcoal and binding. 0.19% (w/v) dextran T-70, final concentrations] as previously described General Methods. Protein concentrations were determined using the (18). In some cases, a hydroxylapatite-based assay for receptor binding method of Lowry ef al. (29), with bovine serum albumin as the standard. of TCDD (19) was used to separate free as well as specifically and nonspecifically bound [3H]TCDD. PSP was labeled with the indicated Radioactivity was measured in a Scintillator 299 from Packard (Downers Grove, IL) in an LKB-Wallac Rackbeta II scintillation spectrometer (Stock concentrations of radioligand at 37Â°C for 30 min, whereafter DCC adsorption was performed at 0-4Â°C (0.1-1% charcoal, final concentra holm, Sweden) with an average counting efficiency of 40%. Safety Precautions. Since TCDD and TCDF are extremely toxic tions, as indicated). All receptor or PSP ligands were dissolved and compounds (8), their use necessitated special handling and disposal added to the incubations in dioxane, the concentration of which never procedures as outlined by Poland ef al. (10). exceeded 1%(v/v). Velocity Sedimentation Analysis. Labeled samples (200 n\) were layered onto linear 5-20% (w/v) or 10-40% (w/v) sucrose gradients in RESULTS buffer (containing 0.4 M KCI in indicated cases), followed by centrifugation By means of gel permeation chromatography of [3H]TCDD- in an SW 60ri rotor in a Beckman L8-M centrifuge at 260,000-320,000 x gavfor 14-18 h to a preset cumulative centrifugal effect (w^) of 1.7 x labeled crude rat ventral prostatic cytosol on Sephacryl S-300 it 1012 rads2/s for TCDD receptor analysis on 10-40% (w/v) sucrose was possible to distinguish between two discrete [3H]TCDD- gradients (17) and 2.15 x 1012 rads2/s for PSP-binding analysis on 5- binding species which both were included into the gel (Fig. 1). 20% (w/v) sucrose gradients. Fractions (s200 Ml/fraction) were collected The first peak of radioactivity with an f?s of approximately 60 A from the bottom of the centrifuge tubes by gravity flow. Cytochrome c was nearly completely abolished by labeling of the crude cytosol (1.7S), bovine serum albumin (4.4S), -y-globulin (6.6S), and catalase in the presence of a 200-fold M excess of TCDF. The 60-Ã• (11.35) were 14C-labeled (20) and used as sedimentation markers. Sedi binding entity was, however, partly obscured by high levels of a mentation velocities of the receptor peaks were calculated according to major [3H]TCDD-binding species eluting in the 25- to 28-Ã•region Martin and Ames (21). of the column. Under these experimental conditions, binding of Gel Permeation Chromatography. Analytical gel permeation chro [3H]TCDD to this entity was nondisplaceable (nonspecific) and matography on 1- x 115-cm columns of Sephacryl S-300 was performed at a flow rate of 5 ml/cm2/h as previously described (18). The columns disturbed the further characterization of the 60-Ã‚ [3H]TCDD- binding species. It was therefore investigated whether heparin- were equilibrated in buffer containing 0.15 M NaCI and 0.02% (w/v) sodium azide. Sephadex G-100 was equilibrated in buffer containing Sepharose chromatography of nonlabeled cytosol could remove 0.02% (w/v) sodium azide and packed into a siliconized column (1.5 x any interfering nonspecific binding. It has previously been estab 87 cm). The flow rate was 14 ml/cm2/h and the sample volumes were lished that the TCDD receptor readily interacts with heparin- approximately 1% of the total column volume. The columns were cali Sepharose both in the presence or absence of ligand (18). It was brated with the following standard proteins: thyroglobulin (86.1 A); ferritin possible to label the material eluted from heparin-Sepharose with

CANCER RESEARCH VOL. 46 FEBRUARY 1986 652

ionic strength. At low ionic strength, it sedimented as an 8-1 OS entity (Fig. 24), whereas at high ionic strength (0.4 M KCI), it -10 - sedimented as a 4-5S entity (Fig. 2B). In the heparin-Sepharose eluates (Fig. 2) as well as in crude cytosol (data not shown), nonspecific [3H]TCDD binding was detectable as a slower sedi- menting entity of 3.6-3.8S under both high- and low-salt condi tions. The fls value of the specific [3H]TCDD-binding entity in prostatic cytosol and its sedimentation coefficient of 4-5S indi cates a highly assymetrical molecule with a molecular weight of approximately 100,000 an f/f0 of 1.7-1.8, and an a/b of 12-15. All of these hydrodynamic properties are indistinguishable from the physicochemical characteristics of the TCDD receptor pres ent in other tissues of the rat (18, 30-32). The concentrations of the specific 60-Ã‚ [3H]TCDD-binding entity varied between 5-20 fmol/mg of cytosolic protein, as estimated by high-resolution gel permeation chromatography on Sephacryl S-300. These obser vations were confirmed by analysis of [3H]TCDD-labeled ventral prostatic cytosols by a hydroxylapatite assay (19) which indi cated TCDD receptor concentrations of approximately 10-12 fmol/mg protein. In this assay, 175 HIM phosphate was sufficient 20 40 60 80 100 to extract all the specifically bound [3H]TCDD which initially had ELUTION VOLUME Imi! Fig. 1. Gel permeation chromatography of rat prostatic cytosol labeled with been immobilized on hydroxylapatite (data not shown). This is in [3H]TCDD. Prostatic cytosol (=10 mg protein/ml) was incubated with 5 nM [3H] TCDD for 2 h at 0-4Â°Cin the absence (O) or presence (â€¢)ofa 200-fold Mexcess agreement with the behavior of the TCDD receptor on hydrox of unlabeled TCDF. Bound and free ligand were separated by adsorption to ylapatite in other tissues, where the TCDD receptor Ã©lÃ»tesfrom prepelletedDCC [final concentration of charcoal, 1.9% (w/v)]. A sample volume of hydroxylapatite at 150-175 rriM phosphate (18,19). Quantitation 1 ml was then chromatographed as described under "Materials and Methods." of specifically bound [3H]TCDD in labeled heparin-Sepharose Fractions (2.0 ml) were collected and assayed for radioactivity. The columns were calibrated with blue dextran 2000 (V0),[uC]glucose (V,)and the following standard eluates yielded concentrations of the specific [3H]TCDD binding proteins: ?, thyroglobulin (86.1 A); 2, ferritin (61.5 Ã€);_3,catalase(51.3 Ã€);4,bovine species of -15-20 fmol/mg protein. However, this value might serum albumin (35.9 A); and 5, cytochrome c (17.9 A). be artificially low due to, e.g., low recovery of the binding entity during the heparin-Sepharose chromatography or difficulties in labeling partially purified TCDD receptor preparations (33). 3 2 1 3 2 1 The hydrodynamic properties of the major [3H]TCDD-binding III 2.0 entity in undiluted cytosol from the rat ventral prostate (fls ~25- 28 A; 3.6-3.8S; cf. above) indicate an apparent molecular weight of 40,000-45,000, an f/f0 of 1.0-1.1, and an a/b of 2-5 which is characteristic of a nearly symmetrical molecule. It has also been shown that [3H]TCDD interacts with a binding species in rat ventral prostatic cytosol which focuses at a pH of about 4.7-5 >â€¢1.0 during isoelectric focusing in polyacrylamide gels and which is distinct from or obscures the TCDD receptor (11). All of these data (summarized in Table 1) closely resemble the physicochem- 0.5

Table 1 SB* Comparisonof physicochemical properties of the major pHJTCDD-binding 10 20 20 top species in rat prostatic cytosol and PSP FRACTION Physicochemicalcharacteristics of the major rat prostatic [3H)TCDD binding Fig. 2. Velocity sedimentationanalysisof [3H]TCDD-labeledheparin-Sepharose species were experimentally determined or calculated as described in "Materials eluates. Nonlabeled prostatic cytosol (5 ml; =50 mg protein) was applied to a and Methods." Properties of PSP were from the literature or in given cases heparin-Sepharose column and then washed with 2-3 column vol of buffer. calculated by the equations used in the text. Retained material was eluted with 1 column vol of buffer containing 0.5 M NaCI and desalted on Sephadex PD-10 columns. The eluates were incubated with 3 nu [3H]TCDD-binding [3H]TCDDfor 2 h at 0-4Â°C,in the absence (O) or presence (â€¢)ofa 200-fold M ParameterS(s-") species3.6-3.8 excess of unlabeled TCDF. Bound and free ligand were separated by DCC 38,000i>-46,000c adsorption [final concentration of charcoal, 1.9% (w/v)]. Aliquots (200 >il)were then M, . 40,000-45,000 layered onto 10-40% (w/v) sucrose gradients prepared in buffer (A) or in buffer fl.(A) 25-28 29" containing 0.4 M KCI (B). The gradients were centrifugea and fractions were 1.3* collected as described under "Materials and Methods." Sedimentation markers fifa 1.1-1.2 Axial ratio 2-5 5-8" were: 1, cytochrome c (1.7S); 2, bovine serum albumin (4.4S); 3, IgG (6.6S); and IsoelectricpointMajor 4.7-5.0"PSP3.7" 4.6-4.9' 4, catalase (11.3S). " Heyns and De Moor (17). 6 Calculated sum of the molecularweight of the different PSP subunits (39). [3H]TCDD and detect specifically bound radioactivity by Sephac- c Forsgrener al. (37). " Calculatedvaluesfrom the sedimentationcoefficient(17)and molecularweight ryl S-300 gel permeation chromatography (data not shown) and (37) (cf. "Materials and Methods"). sucrose gradient analysis (Fig. 2). The velocity sedimentation of eCartstedt-Duke(11). the specific [3H]TCDD-binding entity varied as a function of the ' Heynsef al. (36).

CANCER RESEARCH VOL. 46 FEBRUARY 1986 653

Â¡calcharacteristics of a major secretory protein in the rat ventral prostate, often referred to as PSP (17, 34). In view of these data we examined the binding of various TCDD receptor ligands to crude cytosol as well as to purified PSP.

total cytosolic protein content), suggesting binding of the two ligands to the same site in cytosol. The apparent Ka in this particular experiment was 4.1 nM for [3H]estramustine and 1.9 nw for [3H]TCDD. To further investigate if halogenated PAH interacted with the same class of binding sites in diluted prostatic cytosol as did [3H]estramustine, binding of various concentrations of [3H]estramustine was determined in the absence or presence of 5 or 10 nM TCOF, respectively. TCDF was chosen as a structural ana logue of TCDD because of its more pronounced solubility in water (10). The binding data obtained were analyzed according 10 20 20 top FRACTION to Lineweaver and Burk (35) and were found to be compatible Fig. 3. Velocity sedimentation analysis of purified PSP labeled with [3H]TCDD with a competitive inhibition of [3H]estramustine binding by TCDF (A), or [3H]-3-MC (O) and [3H]B(a)P (â€¢)(B). Purified PSP (a1 mg protein/ml) was (Fig. 5). An apparent Ka of 4.6 nM could be calculated for [3H] incubated with 5 nM [3H]TCDD, 5 nw [3H]-3-MC, or 5 nw [3H]B(a)P at 37Â°Cfor 30 estramustine from the double-reciprocal plot in Fig. 5, which is min. Bound and free ligand were separated by DCC adsorption [final concentration of charcoal, 1% (w/v)]. Aliquots (200 pi) were then layered onto 5-20% (w/v) in good agreement with the value determined by Scatchard plots sucrose gradients prepared in buffer. The gradients were centrifuged and fractions were collected as described under "Materials and Methods". Sedimentation mark (cf. above). Moreover, the number of binding sites determined by double-reciprocal plot analysis in this experiment was approx ers were: 1, myogtobin (2.0S); 2, ovalbumin (3.5S); and 3, bovine serum albumin (4.4S). imately 0.7 nmol/mg cytosolic protein.

CANCER RESEARCH VOL. 46 FEBRUARY 1986 654

Titration of diluted prostatic cytosol with [3H]-3-MC or [3H]- Thus far, it has been shown that the C2 polypeptide (cf. above) B(a)P revealed nonlinear, upward concave curves when apparent can bind steroids (36, 38) and that the CI and C2 chains exhibit equilibrium binding data were analyzed according to Scatchard amino acid sequence homology with each other (34) and with (28) (Fig. 6) indicating at least two classes of binding sites. rabbit uteroglobin (39), whereas the C3 chain exhibits amino acid Interestingly, in addition to low-affinity binding, two straight lines homology with human a2-macroglobulin (43). In the present could be extrapolated from the concave graphs (r s 0.90 and study, [3H]TCDD bound to a saturable binding species in crude 0.92, respectively) indicating the presence of a high-affinity bind cytosol with high affinity (Kâ€žs2 nM). Several lines of evidence ing entity with a Ka of approximately 2 nw for [3H]-3-MC and [3H] support the contention that this binding species in fact represents B(a)P, respectively. In addition, these lines seem to have an PSP: (a) both [3H]estramustine and [3H]TCDD interacted with intercept with the abscissa in the Scatchard plots similar to the the same maximal number of binding sites in crude cytosol as values obtained for [3H]TCDD and [3H]estramustine (cf. Fig. 4). determined by Scatchard plot analysis; (D)double-reciprocal plot analysis according to Lineweaver and Burk (35) showed that TCDF, a structural analogue of TCDD, competitively inhibited DISCUSSION estramustine binding to crude cytosol; (c) the physicochemical properties of the major [3H]TCDD-binding entity in crude cytosol In the present investigation, equilibrium binding studies indi closely resemble those reported for PSP (cf. above); and (d) [3H] cate the presence of a major, saturable, high-affinity binding site TCDD as well as [3H]B(a)P and [3H]-3-MC readily interacted with for [3H]TCDD in diluted cytosol from the rat ventral prostate at purified PSP as assayed by velocity sedimentation analysis. a concentration of about 1 nmol/mg of total cytosolic protein. In Extrapolation of equilibrium binding data calculated according contrast to this observation, hydrodynamic characterization by to Scatchard (28) indicated that both [3H]B(a)P and [3H]-3-MC gel permeation chromatography and sucrose gradient sedimen bound to a similar saturable binding species in diluted cytosol as tation analysis indicates that [3H]TCDD binding to rat prostatic [3H]TCDD and possibly with a similar affinity (Ka s 2 nM). It has cytosol is composed of at least two discrete binding species. previously been shown that [3H]B(a)P and [3H]-3-MC interact The major fraction of bound [3H]TCDD was associated with a with a 4S, high-capacity, high-affinity carcinogen-binding protein symmetrical molecule of 3.6-3.8S, an Rs of 25-28 A, and a in rat liver cytosol with very similar affinities (Ka = 2.5 and 2.8 molecular weight of approximately 40,000-45,000. These phys- nM, respectively) (44,45). A similar protein binding [3H]B(a)P with icochemical characteristics agree well with the properties of PSP, a Ka of 1.8 nM has recently been described in a mouse embryo a protein purified by several laboratories from the rat ventral cell line (46). prostate. (36-38). PSP is a tetrameric glycoprotein with an The mechanism of induction of cytochrome P-450c in the rat isoelectric point of about 4.6-4.9 (36) consisting of two subunits, ventral prostate is intriguing. There exists an increasing amount one containing the two different polypeptides termed CI (M, ~ of data on the induction of AH H activity and cytochrome P-450c 10,200) and C3 (M, ~ 8,600) and the other containing C2 (M, ~ in the rat ventral prostate (3-7). However, no TCDD receptor 10,500) and C3 (34, 39). The carbohydrate content of PSP has has been observed in this tissue in the rat (11,12), whereas the been estimated to be 3.2% (36). The most prominent functional receptor has been detected in mouse prostatic cytosol4 (12) characteristic of PSP appears to be steroid binding (17, 36). suggesting a nonclassical nonreceptor mediated induction pro Androgens induce PSP synthesis (40, 41) and bind to PSP with cess of cytochrome P-450c in the rat prostate. Interestingly, low affinity (Ka ~1 pM) (17, 34). Moreover, it is known that analytical gel permeation chromatography of [3H]TCDD-labeled estradici mustard derivatives which are used in treating prostatic crude cytosol also indicated binding of [3H]TCDD to a discrete carcinoma (42) bind to PSP with high affinity (Ka~10-30 nw) (26, component in cytosol with an fls of ~60 A. Moreover, prechro- 27, 37). The biological function of PSP is presently not known. matography of unlabeled cytosol on heparin-Sepharose revealed the presence of a specific [3H]TCDD binding species which sedimented at 8-1 OS under low-ionic strength conditions and 4-5S under high-ionic strength conditions and which was hardly distinguishable in crude cytosol. This salt dependent sedimen CL O.It - tation shift is characteristic of the TCDD receptor Â¡nseveral tissues (18,30) and of steroid hormone receptors in general (47). Furthermore, the physicochemical characteristics of this binding species, i.e., sedimentation coefficients, fls, and molecular weight 0.2 - are identical to those observed in rat liver cytosol for the TCDD receptor (18, 33). In view of these data, we believe that this particular binding species represents the TCDD receptor and that its detection in crude cytosol is severely hampered by the high abundance of high-affinity [3H]TCDD-binding to PSP. In 0.5 1.0 1.5 spite of the abundance of the major [3H]TCDD-binding species, BOUND I n M I Fig. 6. Scatchardanalysisof the binding of [3H]B(a)Pand[3H]-3-MCto prostatic analysis of crude cytosol by gel permeation chromatography cytosol. Cytosol (si .3 Â»igprotein/mlbuffer containing0.1% gelatin)was incubated indicates the presence of the receptor. Several explanations are at 37Â°Cfor 30 min with increasingconcentrations (0-20 nM)of [3H]B(a)P(D) and |3H|-3-MC (O) before binding was assayed as described under "Materials and conceivable for this observation, e.g. (a) a subsaturating concen Methods" and in the legend to Fig. 4. Points, mean of duplicate determinations. tration of [3H]TCDD with regard to binding to the major binding The broken lines were extrapolated from the graphs by linear regression analysis (r ~ 0.90-0.92) to obtain tentative Kasfor a possible high-affinitycomponent. 4P. Soderkvist, L. Poellinger,and J-Ã•.Gustafsson,unpublishedresults.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. CARCINOGEN-BINDING PROTEINS IN THE RAT PROSTATE species and (b) differences in affinities of the TCDD receptor and 2. Lernen, R. A., Lee, J. S., Wagoner, J. K., and Bleijer, H. P. Cancer mortality among cadmium production workers. Ann. NY Acad. Sci., 277: 273-279, the major binding species, respectively. For instance, the rat 1976. hepatic TCDD receptor has a Ka for [3H]TCDD of 0.2-0.8 nM 3. SÃ¶derkvist, P., ToftgÃ¢rd, R., and Gustafsson, J-Ã€.Induction of cytochrome P- (19). Moreover, no information is available at the present on the 450 related metabolic activities in the rat ventral prostate. Toxico). Lett (Amst.), dissociation rates of [3H]TCDD from either the major binding 70:61-69,1982. 4. Haaparanta, T., Glaumann, H., and Gustafsson, J-Ã‚. Induction of cytochrome species or the TCDD receptor. Gel permeation chromatography P-450 dependent reactions in the rat ventral prostate by /3-naphthoflavone and analysis on Sephacryl S-300 was carried out during approxi 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicology, 29:61-75,1983. 5. Haaparanta, T., Halpert, J., Glaumann, H., and Gustafsson, J-Ã€.Immunochem- mately 24 h. Therefore, the loss of bound ligand due to dissocia ical detection and quantification of microsomal cytochrome P-450 and reduced tion and association to the gel matrix might be considerable. nicotinamide adenine dinucleotide phosphatercytochrome P-450 reducÃasein the rat ventral prostate. Cancer Res., 43: 5131-5137,1983. The pronounced abundance of PSP in prostatic epithelial cells 6. SÃ¶derkvist, P., Busk, L., ToftgÃ¢rd,R., and Gustafsson, J-Ã‚. Mtabolic activation (S20% of the total protein content in cytosol) and in seminal fluid of promutagens, detectable in Ames' Salmonella assay, by 5000 x g super (17) confers a potentially high binding capacity for chemical natant of rat ventral prostate. Chem.-Biol. Interact., 46: 151-163,1983. 7. Haaparanta, T., NorgÃ rd, M., Haglund, L., Glaumann, H., and Gustafsson, J- carcinogens like B(a)P and 3-MC as well as for toxic substances Ã‚. Immunohistochemical localization of cytochrome P-450 and reduced nico such as chlorinated dibenzodioxins and dibenzofurans and might tinamide adenine dinucleotide phosphate:cytochrome P-450 reducÃase in the cause the accumulation of these and possibly related substances ral ventral prostate. Cancer Res., 45: 1259-1262,1985. 8. Poland, A., and Knutson, J. C. 2,3,7,8-Tetrachlorodibenzo-p-dioxinand related in the prostate. High-capacity binding of the chlorinated hydro halogenated hydrocarbons: examination of the mechanism of toxicity. Annu. carbon insecticides dieldrin and o,p'-dichlorodiphenyltrichloro- Rev. Pharamcol. Toxico).. 22: 517-554,1982. ethane to a 3.5S androgen-binding protein in cytosol from the 9. Poland, A., and Glover, E. Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. rat ventral prostate has previously been described (48). The Mol. Pharmacol., 70: 349-359, 1974. properties of this protein are similar to those of PSP (cf. Table 10. Poland, A., Glover, E., and Kende, A. S. Stereospecific high-affinity binding of 1). In vivo studies have shown that o,p'-dichlorodiphenyltri- 2,3,7,8-tetrachlorodioenzo-p-dioxin by hepatic cytosol. Evidence that the bind ing species is a receptor for induction of aryl hydrocarbon hydroxylase. J. Biol. chloroethane accumulates in the mouse prostate (49). A 3-4S Chem. 251: 4936-4946,1976. carcinogen- and TCDD-binding protein has been observed in 11. Carlstedt-Duke, J. Tissue distribution of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Cancer Res., 39: 3172-3176,1979. mouse prostatic cytosol, albeit at lower concentrations as com 12. Mason, M. E., and Okey, A. B. Cytosolic and nuclear binding of 2,3,7,8- pared to in rat prostatic cytosol4 (12). Furthermore, in vivo uptake tetrachlorodibenzo-p-dioxin to the Ah receptor in extrahepatic tissues of rats and secretion of 3-MC by the rat and dog prostates has been and mice. Eur. J. Biochem., 723: 209-215,1982. 13. McKeehan, W. L., and Fast, D. The major androgen-dependent protein in rat demonstrated (50). Thus, it is conceivable that PSP is of rele ventral prostate binds polycyclic aromatic hydrocarbons. Cell. Biol. Int. Rep., vance for the tissue-specific accumulation and secretion of struc 5:2,1981. turally related PAH. 14. Haaparanta, T., Pousette, A, Hogberg, B., Gustafsson, J-Ã€.,and Glaumann, H. Fractionation of the rat ventral prostate with respect to isolation and It is interesting that a secretory protein exhibiting amino acid exocytosis of the prostatic secretion protein. Biochim. Biophys. Acta, 776: 79- homology with PSP, uteroglobin (39) (cf. above), also binds 93,1982. steroids with a similarly low affinity as compared to PSP (Ka s 1 15. Heyns, W., Bossyns, D., Peelers, B., and Rombauts, W. Study of a proline- nM) and polychlorinated biphenyl congeners such as 4,4'- rich polypeptide bound to the prostatic binding protein of rat ventral prostate. bis(methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl with high affin J. Biol. Chem., 257: 7407-7413,1982. 16. Ichii, S. 5a-Dihydrotestosterone binding protein in rat ventral prostate: purifi ity (Ka = 2.5-15 nM) (51). The physiological roles of either PSP cation, nuclear incorporation and subnuclear localization. Endocrino). Jpn., 22: or uteroglobin or the toxicological implications of the PAH-protein 433-477, 1975. 17. Heyns, W., and De Moor, P. Prostatic binding protein: a steroid-binding protein interaction are presently not understood. Furthermore, it is not secreted by rat prostate. Eur. J. Biochem., 78: 221-230,1977. known if high-capacity binding of PAH by e.g. PSP is of impor 18. Poellinger, L., Lund, J. Gillner, M., Hansson, L-A., and Gustafsson, J-Ã€. tance for the mechanism of induction of cytochrome P-450c. Physicochemical characterization of specific and nonspecific polycyclic hydro carbon binders in rat and mouse liver cytosol. J. Biol. Chem, 258: 13535- However, it has been suggested that carcinogen-binding mole 13542,1983. cules in rat liver may participate in the microsomal metabolism 19. Poellinger, L., Lund, J., Dahlberg, E., and Gustafsson, J-Ã€.A hydroxylapatite microassay for receptor binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3- of PAH as intracellular transport proteins (52, 53). Unfortunately, methylcholanthrene in various target tissues. Anal. Biochem., 744: 371-384, these molecules have not yet been identified or purified. It is 1985. conceivable that PSP might be functionally similar to such a 20. Rice, R. H., and Means, G. E. Radioactive labeling of proteins in vitro. J. Biol. Chem., 246: 831-832,1971. molecule in rat liver. Therefore, the rat ventral prostate might 21. Martin, R. G., and Ames, B. N. A method for determining the sedimentation present an attractive model for further studies of the mechanism behavior or enzymes: application to protein mixtures. J. Biol. Chem., 236: of induction of cytochrome P-450c and the microsomal metab 1372-1379,1961. 22. Siegel, L. M., and Monty, K. J. Determinations of molecular weights and olism of PAH. frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hy- droxylamine reducÃase.Biochim. Biophys. Acta, 772: 346-362,1966. ACKNOWLEDGMENTS 23. Schachman, H. K. Ultracentrifugation in Biochemistry, p. 239. New York: Academic Press, Inc., 1959. We thank Dr. A. Poland (University of Wisconsin, Madison, Wl) for providing 24. Sherman, M. R., Tuazon, F. B., and Miller, L. K. Estrogen receptor cleavage [3H]TCDD, Dr. C. Rappe (Univerity of UmeÃ ,Sweden) for unlabeled TCDF and Dr. and plasminogen activation by enzymes in human breast tumor cytosol. B. Forsgren (AB Leo Research Laboratories, Helsingborg, Sweden) for [3H]estra- Endocrinology, 706:1715-1727,1980. mustine. Dr. J. Lund (Karolinska Institute, Stockholm, Sweden) is gratefully ac 25. Edelnoch, H. The properties of thyroglobulin. I. The effects of alkali. J. Biol. knowledged for fruitful discussions throughout the study. Chem., 235: 1326-1334,1960. 26. Forsgren, B., Gustafsson, J-Ã€., Pousette A., and Hogberg, B. Binding char acteristics of a major protein in rat ventral prostate cytosol that interacts with REFERENCES estramustine, a nitrogen derivative of 17/3-estradiol. Cancer Res., 39: 5155- 5164, 1979. 1. Goldsmith, D. F., Smith, A. H., and McMichael, A. J. A case-control study of 27. Heyns, W., and Bossyns, D. A. Comparative study of estramustine and prostate cancer within a cohort of rubber and tire workers. J. Occup. Med., pregnenolone binding to prostatic binding protein: evidence for subunit coop- 22:533-541,1980. erativity. J. Steroid Biochem., 79: 1689-1694,1983.

CANCER RESEARCH VOL. 46 FEBRUARY 1986

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28. Scatchard, G. The attraction of proteins for small moleculesand ions. Ann. NY prostatic C3 gene transfectedinto mousemammarytumor cells. Cell,32:495- Acad. Sci., 5/: 660-672,1949. 502,1983. 29. Lowry, O. H., Rosebrough, N. J., Fair, A. L, and Randall, R. J. Protein 42. Jonsson, G., Hogberg,B., and Madsen, P. O. Treatment of advancedprostatic measurementwith folin phenol reagent. J. Biol. Chem., 793: 265-275,1951. carcinoma with estramustine phosphate. Scand. J. Urol. Nephrol., 77: 231- 30. Lund, J., Kurt, R. N., Poellinger,L., and J-Ã€.Gustafsson.Cytosolic and nuclear 238, 1977. binding proteins for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat thymus. 43. Baker, M. E. Amino acid homology between the C3 chain of rat prostatic Biochim. Biophys. Acta, 776: 16-23,1982. steroid binding protein and human alphaj-macroglobulin. Biochem. Biophys. 31. Poellinger,L., Kurt, R. N., Lund, J., Gillner, M., Cartstedt-Duke,J., Hogberg, Res. Commun., 722: 662-667, 1984. B., and Gustafsson, J-Ã€.High-affinitybinding of 2,3,7,8-tetrachlorodibenzo-p- 44. Holder, G. M., Tiemey, B., and Bresnick, E. Nuclear uptake and subsequent nuclearmetabolismof benzo(a)pyrenecomplexedto cytosolic proteins. Cancer dioxin in cell nucleifrom rat liver. Biochim. Biophys. Acta, 714: 516-523,1982. Res., 47. 4408-4414, 1981. 32. Poellinger, L., Lund, J., Gillner, M., and Gustafsson, J-Ã•.The receptor for 45. Tiemey, B., Weaver, D., Heintz, N. H., Schaeffer, W. I., and Bresnick, E. The 2,3,7,8-tetrachlorodibenzo-p-dioxin:similaritiesand dissimilaritieswith steroid identity and nuclear uptake of a cytosolic binding protein for 3-methylcholan- hormone receptors. In: V. K. Moudgil (ed.). Molecular Mechanismof Steroid threne. Arch. Biochem. Biophys., 200: 513-523, 1980. HormoneAction, pp. 755-790. New York: Walter de Gruyter, 1985. 46. Zytkovicz, T. H. Identificationand characterization of a high-affinity saturable 33. Poellinger,L., and Gullberg, D. Characterizationof the hydrophobic properties binding protein for the carcinogen benzo(a)pyrene.Cancer Res., 42: 4387- of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol., 27: 4393, 1982. 271-276, 1985. 47. Sherman, M. R., and Stevens, J. Structure of mammaliansteroid receptors: 34. Parker, M., Needham,M., and White, R. Prostatic steroid bindingprotein: gene evolving concepts and methodologicaldevelopments.Annu. Rev. Physiol.46: duplication and steroid binding. Nature (Lond)., 298: 92-94, 1982. 83-105,1984. 35. Lineweaver. H., and Burk, D. The determination of enzyme dissociation con 48. Wakeling,A. E., and Visek, W. J. Insecticideinhibition of Sn-dihydrotestoster- stants. J. Am. Chem. Soc., 56: 658-666, 1934. one binding in the rat ventral prostate. Science (Wash. DC), 787: 659-661, 36. Heyns,W., Peelers, B., Mous, J., Rombouts, W., and De Moor, P. Purification 1973. and characterisation of prostatic binding protein and its subunits. Eur. J. 49. Smith, M. T., Thomas, J. A., Smith, C. G., Mawhinny, M. G., and Lloyd, J. W. Biochem.,09: 181-186,1978. Effects of DDT on radioactive uptake from testosterone-1,2-[3H] by mouse 37. Forsgren, B., Bjork, P., CarlstrÃ¶m,K., Gustafsson, J-Ã•.,Pousette, Ã€.,and prostate glands. Toxico!. Appi. Pharmacol.,23:159-164,1972. 50. Smith, E. R., and Hagopian,M. The uptake and secretion of 3-methylcholan- HÃ¶gberg,B.Purificationand distribution of a major protein in rat prostate that binds estramustine, a nitrogen mustard derivative of estradiol-17|a.Proc. Nati. threne by the prostate glands of rat and dog. J. Nati. Cancer Inst. 59: 119- Acad. Sci. USA, 76: 3149-3153, 1979. 122,1977. 38. Chen, C., Schilling, K., Hiipakka, A., Huang, I-Y., and Liao, S. Prostate a- 51. Lund, J., Brandt, I., Poellinger, L., Bergman, A., Klasson-Wehler, E., and Gustafsson, J-Ã€.Targetcells for the polychlorinated biphenyl metabolite 4,4- protein: isolation and characterization of the polypeptide components and bis(methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl:characterizationof high affin cholesterol binding. J. Biol. Chem., 257:116-121, 1982. ity bindingin rat and mouse lungcytosol. Mol. Pharmacol.,27:314-323,1985. 39. Baker, M. E. Amino acid homology between rat prostatic steroid binding 52. Hanson-Painton,0., Griffin, M. J., and Tang, J. Involvement of a cytosolic protein and rabbit uteroglobin. Biochem. Biophys. Res. Commun., 774: 325- carrier protein fraction in the microsomalmetabolism of benzo(a)pyrenein rat 330, 1983. liver. Cancer Res., 43: 4198-4206,1983. 40. Page, M. J., and Parker, M. G. Effect of androgen on the transcription of rat 53. Collins,S., and Marietta, M. A. Carcinogenbindingprotein. Highaffinity binding prostatic binding protein genes. Mol. Cell. Endocr., 27: 343-355,1982. sites for benzo(a)pyrenein mouse liver distinct from the Ah receptor. Mol. 41. Page, M. J., and Parker, M. G. Androgen-regulatedexpression of a cloned rat Pharmacol.,26: 353-359, 1984.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. Carcinogen-binding Proteins in the Rat Ventral Prostate: Specific and Nonspecific High-Affinity Binding Sites for Benzo( a)pyrene, 3-Methylcholanthrene, and 2,3,7,8-Tetrachlorodibenzo- p-dioxin

Peter Söderkvist, Lorenz Poellinger and Jan-Åke Gustafsson

Cancer Res 1986;46:651-657.

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